A team of researchers led by scientists at Iowa State University has been awarded nearly $3 million to use microarray and next-generation sequencing technology to identify functional structural diversity among maize haplotypes.

The US National Science Foundation awarded the researchers $1.5 million on March 15 and will dole out $1.5 million more when the first year of the grant expires on Feb. 29, 2012, according to principal investigator Patrick Schnable, director of ISU's Center for Plant Genomics.

Schnable told BioArray News last week that his team will use Illumina sequencing and Roche NimbleGen comparative genomic-hybridization arrays to study the functional significance of genetic variation identified in 26 lines of maize in comparison to the reference genome. He added that he is still "not clear" how the researchers will "balance between" the two technologies as the study progresses.

Co-investigators listed on the project are Brent Buckner, a professor of biology at Truman State University; Daniel Nettleton, a biostatistician at ISU; and Carolyn Lawrence, an ISU-based US Department of Agriculture scientist.

Their work builds on earlier studies that centered on annotating a popular line of maize called B73, which has served as a reference genome for the maize community. In November 2009, Schnable and other researchers published a paper in PLoS Genetics that showed that certain genes in the reference genome do not appear in other maize lines (BAN 1/26/2010).

"Using CGH we detected that there were some genes missing in some haplotypes, some inbred lines, as compared to the reference genome," said Schnable, who performed a follow-up, resequencing-based study that supported the original findings.

Schnable said those studies showed that some of the missing genes do in fact impose a phenotype.

"Once we knew genes were missing, it wasn't surprising to me that we would find phenotypes," he said. "But it is very satisfying that we are starting to find that."

Wild Ancestor

As part of the new NSF-funded work, Schnable and his colleagues will look at 26 distinct maize lines to identify genetic variations and determine whether they have functional significance.

"Yes, these genes are missing, but do they matter? That is one of the major foci of this NSF award: to try and test that," he said, adding that it's a "very important question for us and for the seed companies."

They will use Illumina technology to sequence the transcriptomes of the 26 lines and align the data to the B73 reference genome. Schnable said the lines were selected to "sample the genetic diversity of maize."

All sequencing data will be deposited into the National Center for Biotechnology Information's GenBank database, and all project data will be made available to the maize community via several existing databases, including MaizeGDB, Gramene, and Panzea, the researchers said.

The team will also study the genetic differences between maize's wild ancestor, called teosinte, and modern-day crop lines. According to Schnable, domesticated maize was created from teosinte about 10,000 years ago in Central America.

"We are asking, 'Are there genes in teosinte that are missing from maize?'" Schnable said.
The group has already found some genes in teosinte that do not appear in the reference genome and will use Roche NimbleGen CGH arrays to "ask if those genes are missing from all maize lines or just some maize lines," he said.

Ultimately, the team hopes to assemble a "Zeanome" — a "near-complete set" of genes present in B73, other maize lines, and teosinte — that could enable researchers to look for structural variations in other lines, according to the grant abstract.

The team has three hypotheses about structural variation in maize: that it helped early farmers domesticate maize, led to the "extraordinary phenotypic diversity and plasticity" of the crop, and contributed to the success of long-term selection.

The studies will also try to look for data that could benefit other parts of the maize-crop industry. For instance, findings suggesting that gene copy-number changes contribute to genetic gain would be "transformative to the breeding industry," the researchers claimed in the abstract.

"To help adapt crops to climate change it may be desirable to reintroduce into breeding germplasm stress-resistance genes and genetic diversity [that were] inadvertently lost during domestication," they said. Better understanding of such variants could help breeders improve hybrids, the researchers anticipated.

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'Sequencing is Better'

Though CGH will play a role in the studies, Schnable said he is considering using sequencing rather than arrays to profile gene expression and genotype samples.

"CGH is here, it works great, and we are using it right now in the project, and we will use it," he said. "What I am still exploring is if we can do some of the genotyping work by sequencing."

It wouldn't be a surprising move as Schnable is an early adopter of next-generation sequencing. The first research projects performed in his lab on next-gen sequencers resulted in papers published as early as 2007, less than a year after Illumina began selling its first line of next-gen sequencers.

And last year, Schnable became the managing partner of Data2Bio, a firm that helps clients design studies based on next-generation sequencing and analyze the data.

It was also at around that time that Schnable removed gene expression microarrays from the menu at the Center for Plant Genomics.

"I shut my [gene expression array] facility down, and now recommend that everybody go to RNA-seq for that question," he said. "There are no doubt niche questions for which microarrays are still the right technology, but for the discovery stuff we are doing it's clear that sequencing methods are better."

The center is still set up to perform CGH studies and Schnable said he does not plan to stop. CGH is "going to be around for quite a while," he said.

He also said that DNA sequencing will become more popular as prices continue to fall and better data-analysis tools become available. Though Illumina last year began selling a MaizeSNP50 BeadChip for genotyping projects (BAN 1/19/2010), Schnable said it is unlikely that higher-density genotyping chips will become available, even though they are currently made for other organisms, such as bovines.

"I do not think that will happen in maize," Schnable said. "The maize chip is certainly getting used and is powerful and useful, but … hybridization-based methods aren't going to compete" with DNA sequencing.

Like most next-gen-sequencing users, he conceded that the technology presents data-analysis challenges, but maintained that the "data-analysis pipelines will all get built" and make analysis as "easy as turning a key."